JP2008545136A - Nucleic acid biosensor using photoelectrochemical amplification - Google Patents

Abstract

The present invention relates to electrode systems, methods, devices, and chips for ultrasensitive detection and quantification of nucleic acids using photoelectrochemical amplification. Upon hybridization of the target nucleic acid to the nucleic acid capture probe, the optical reporter containing the threaded bis-intercalator selectively binds to the double stranded nucleic acid. The stability and reversibility of the photoreporter binding activity provides ultrasensitive detection of nucleic acid hybridization events.

Description

TECHNICAL FIELD The present invention relates to an electrode system, method, apparatus and chip for ultrasensitive detection and quantification of nucleic acids using photoelectrochemical amplification by binding a photoreporter to a double-stranded nucleic acid analyte complex. .

BACKGROUND OF THE INVENTION The use of biosensors to test conventional gene expression involves the use of labeled cDNA or cRNA targets derived from experimental sample mRNA that hybridize to nucleic acid capture probes bound to a solid support. ing. By monitoring the amount of label associated with each hybridization event, it was possible to infer the abundance of each mRNA species indicated. Although hybridization is sometimes used to detect and quantify nucleic acids, the combination of the miniaturization of this technique and the enormous and increasing amount of sequence information prohibits the scale at which gene expression can be studied. Enlarged.

Great for the development of nucleic acid biosensors based on the immobilization of short oligonucleotide capture probes on a solid phase that can be used as a biorecognition element during subsequent hybridization with target sample nucleic acids over the past decade There is progress. The most common method is still relying on fluorescently conjugated target nucleic acids ¹ .

However, the appropriate sensitivity of detection of low copy number genes remains problematic in current fluorescence-based microarray technology applications. Modern fluorescence microarray assays are performed in combination with labeling methods such as liquid phase (off-chip) pre-amplification and / or polymerase chain reaction (PCR) ² . However, PCR amplification not only extends assay time, but often introduces contaminating amplicon species. In addition, the genes are not shown at a relatively proportional level in the final PCR product compared to the first sample due to selective and non-linear target amplification ³ . In addition, incomplete denaturation of the nucleic acid secondary structure during cDNA synthesis also impairs polymerase activity, resulting in a truncated cDNA copy of the target gene. Furthermore, the PCR-based pre-amplification step is a limited application in analyzing high complexity nucleic acids because the products of such PCR interfere with each other, thereby resulting in a loss of amplification efficiency and specificity. There is ⁴ . Off-chip targeted pre-amplification also significantly increases the cost of biosensor approaches and sometimes leads to sequence-dependent quantitative bias.

To address these technical difficulties, several, such as rolling circle amplification ⁴ , branched DNA technology ⁵ , catalytic reporter binding ⁶ , dendritic tag ⁷ , enzymatic amplification ^8,9 , and chemical amplification ^10,11 An on-chip amplification strategy has been proposed. Among these, amplification strategies linked to electrical transduction methods are simplified for nucleic acid assays due to the inherent miniaturization of electronic devices and their compatibility with advanced semiconductor technologies. With the greatest potential to provide an accurate and inexpensive platform.

  The present invention shows that threaded bis-intercalator PIND-Ru-PIND functions as a highly sensitive, highly stable and highly selective light reporter, enabling photoelectrochemical detection of nucleic acid hybridization events It is based on the surprising and unexpected knowledge of the present inventors that it can be achieved.

SUMMARY OF THE INVENTION According to a first aspect of the present invention, an electrode system is provided, the electrode system comprising:
(a) working electrode;
(b) a nucleic acid capture probe linked to a working electrode, comprising a sequence complementary to the sequence of the target nucleic acid; and
(c) Including sewn-in type PIND-Ru-PIND screw intercalator,
The target nucleic acid hybridizes with the nucleic acid capture probe to form a double stranded nucleic acid complex into which the threaded PIND-Ru-PIND bis-intercalator intercalates. The amount of threaded PIND-Ru-PIND intervened indicates the amount of target nucleic acid. This electrode system can detect and / or quantify the target nucleic acid.

  The electrode system can further include a support having a working electrode disposed thereon.

  The working electrode can include an array of nucleic acid capture probes.

  The working electrode can include at least one of diamond, glassy carbon, gold, graphite, indium tin oxide, platinum or silicon. The working electrode can include indium tin oxide.

  The nucleic acid capture probe can include DNA. In addition or alternatively, the nucleic acid capture probe can comprise RNA. The nucleic acid capture probe can be bound to the working electrode.

  The target nucleic acid can include DNA. The DNA can include cDNA. In addition or alternatively, the target nucleic acid can comprise RNA. The RNA can include cRNA.

  The electrode system can further include a reference electrode and a counter electrode.

In one embodiment of the first aspect, the intercalation of the double-stranded nucleic acid complex and the threaded PIND-Ru-PIND bis-intercalator is 2 from the threaded PIND-Ru-PIND bis-intercalator. Involved in the intercalation of two naphthalene diimide groups and double-stranded nucleic acid complexes, resulting in the divalent positive of the phosphate group of the double-stranded nucleic acid complex and the threaded PIND-Ru-PIND bis-intercalator An ion pair can be formed between the ionic (bicationic) Ru (bpy) ₂ ²⁺ group. The stable adduct formed thereby produces a photoelectrochemical reaction upon exposure to a light source and can produce a photocurrent action spectrum in the 400-600 nm range, particularly 490 nm. The application of irradiation cycles can cause photocharge and discharge currents that increase substantially linearly up to 10 ³ cycles as the number of irradiation cycles increases. The stability and sensitivity of intervening threaded PIND-Ru-PIND bis-intercalators can provide detection of target nucleic acids up to 1 × 10 ⁻¹⁶ M target nucleic acid dilutions.

According to a second aspect of the present invention, a method for detecting a target nucleic acid is provided, which comprises:
(a) hybridizing a target nucleic acid with a nucleic acid capture probe linked to a working electrode to form a double-stranded nucleic acid complex;
(b) intervening a threaded PIND-Ru-PIND bis-intercalator in the double-stranded nucleic acid complex;
(c) irradiating the working electrode with light comprising at least one wavelength that photoactivates the PIND-Ru-PIND bis-intercalator;
(d) applying a potential to the working electrode; and
(e) including a step of obtaining a photocurrent action spectrum.

  The amount of target nucleic acid can be determined from the photocurrent action spectrum.

According to a third aspect of the present invention, a method for quantifying a target nucleic acid is provided, the method comprising:
(a) hybridizing a target nucleic acid with a nucleic acid capture probe linked to a working electrode to form a double-stranded nucleic acid complex;
(b) intervening a threaded PIND-Ru-PIND bis-intercalator in the double-stranded nucleic acid complex;
(c) irradiating the working electrode with light comprising at least one wavelength that photoactivates the PIND-Ru-PIND bis-intercalator;
(d) applying a potential to the working electrode; and
(e) including a step of obtaining a photocurrent action spectrum.

  The amount of target nucleic acid can be determined from the photocurrent action spectrum.

According to a fourth aspect of the present invention, an apparatus for detecting a target nucleic acid is provided, the apparatus comprising:
(a) the electrode system of the first aspect;
(b) means for irradiating the working electrode of the electrode system;
(c) means for applying a potential to the working electrode; and
(d) includes means for obtaining a photocurrent action spectrum.

  In use, irradiation of the working electrode of the electrode system by means of irradiating the working electrode, and application of potential to the working electrode by means of applying potential to the working electrode is a photocurrent action obtained by means of obtaining a photocurrent action spectrum. Produces a spectrum. The amount of target nucleic acid can be determined from the photocurrent action spectrum.

  The apparatus can further include a support on which the working electrode is disposed.

  The device can optionally further include a data capture device. The data capture device can provide a means for applying a potential to the working electrode. The data capture device can provide a means for obtaining a photocurrent action spectrum of the working electrode.

  The apparatus can further include a fluid system. The fluid system can provide a means for delivering the target nucleic acid to the electrode system.

  This device may further provide a temperature control device. The temperature control device can be used in combination with a fluid system.

  The apparatus can further include an optical scanner or detector. An optical scanner or detector can receive data from the electrode system. This data can be a sample identifier. In addition or alternatively, the data may be fluorescence data.

According to a fifth aspect of the present invention, there is provided an apparatus for quantifying a target nucleic acid, the apparatus comprising:
(a) the electrode system of the first aspect;
(b) means for irradiating the working electrode of the electrode system;
(c) means for applying a potential to the working electrode; and
(d) includes means for obtaining a photocurrent action spectrum.

  In use, irradiation of the working electrode by means of irradiating the working electrode of the electrode system and application of potential to the working electrode by means of applying potential to the working electrode generates a photocurrent action spectrum, which Obtained by means of obtaining a spectrum. The amount of target nucleic acid can be determined from the photocurrent action spectrum.

  The apparatus can further include a support on which the working electrode is disposed.

  The device can optionally further include a data capture device. The data capture device can provide a means for applying a potential to the working electrode. The data capture device can provide a means for obtaining a photocurrent action spectrum of the working electrode.

  The apparatus can further include a fluid system. The fluid system can provide a means for delivering the target nucleic acid to the electrode system.

  This device may further provide a temperature control device. The temperature control device can be used in combination with a fluid system.

  The apparatus can further include an optical scanner or detector. An optical scanner or detector can receive data from the electrode system. This data can be a sample identifier. In addition or alternatively, the data may be fluorescence data.

According to a sixth aspect of the present invention, a biosensor chip is provided, which
(a) chip;
(b) at least one working electrode disposed on the chip;
(c) a nucleic acid capture probe linked to a working electrode, comprising a sequence complementary to the sequence of the target nucleic acid; and
(d) Includes a stitched-type PIND-Ru-PIND screw-intercalator.

  The tip can include a working electrode.

  The working electrode can include an array of nucleic acid capture probes.

  The working electrode can include at least one of diamond, glassy carbon, gold, graphite, indium tin oxide, platinum or silicon. The working electrode can include indium tin oxide.

  The nucleic acid capture probe can include DNA. In addition or alternatively, the nucleic acid capture probe can comprise RNA.

  The target nucleic acid can include DNA. The DNA can include cDNA. In addition or alternatively, the target nucleic acid can comprise RNA. The RNA can include cRNA.

Definitions In the context of this specification, the term “comprising” means “mostly, but not necessarily, simply including”. Furthermore, variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly modified meanings.

  The term “primer” as used herein is used interchangeably with the term “oligonucleotide”. The term “primer” refers to a single stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis. An “oligonucleotide” is a single-stranded nucleic acid typically from 2 to about 500 bases in length. The exact length of the oligonucleotide will vary according to the particular application, but is typically considered in the range of 15-30 nucleotides. The oligonucleotide need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template.

  As used herein, the term “hybridize” means in the context of a nucleic acid to form a base pair between the complementary regions of two nucleic acids that do not initially form a pair.

  The term “chip” as used herein is used interchangeably with the term “array” or “microarray” and refers to a device that includes or has a working electrode disposed thereon. Typically, the nucleic acid capture probe is linked to the working electrode.

BEST MODE FOR CARRYING OUT THE INVENTION The threaded bis-intercalator PIND-Ru-PIND was used in experiments to demonstrate ultrasensitive unlabeled detection of nucleic acid hybridization events.
PIND-Ru-PIND:
PIND = N, N'-bis (3-propyl-imidazole) -1,4,5,8-naphthalenediimide
Ru = Ru (bpy) ₂ ²⁺ (bpy = 2,2'-bipyridine)
Compared to direct voltammetric detection, a remarkable sensitivity enhancement was realized. Light electrochemical signal is observed when there is a small nucleic acid such fmoles (10 ^-15 M) weight, this represents a 10 ⁴ fold increase in the sensitivity of nucleic acid detection over conventional techniques. The present invention thus provides an improved method of application of hybridization-based nucleic acid biosensors.

  FIG. 1 illustrates one embodiment of the present invention. The system (100) includes a support (110), a working electrode (120) disposed on the support (110), a data capture device (160), a light source (170), and optionally a counter electrode (180), see It includes an electrode (190), a fluid system (200), a temperature controller (210) and an optical scanner or detector (220). The nucleic acid capture probe (130) is linked to the working electrode (120) and includes a sequence that is complementary to the sequence of the target nucleic acid (140). Upon hybridization of the target nucleic acid (140) and the nucleic acid capture probe (130) to form a double-stranded nucleic acid complex, the complex has a threaded PIND-Ru-PIND bis-intercalator (150). The amount of intervening and intervening PIND-Ru-PIND (150) indicates the amount of target nucleic acid (140).

  This support (110) can comprise any material and can be any shape, size, dimension, density and / or conductivity as required to perform the present invention. In some embodiments, the support (110) and / or working electrode (120) is combined with an optical scanner or detector (220) at a position on the working electrode (120) that is illuminated by the light source (170). Includes means for determining. For example, an identifier such as a grid or other reference mark or index for determining the irradiation position may be provided. Such identifiers can be applied to the support (110) and / or the working electrode (120) by any method known in the art that enables the performance of the present invention, which may be physical vapor deposition or This includes but is not limited to chemical vapor deposition, lithography, ion assisted or electrochemical etching or electroplating. In another embodiment, when the working electrode (120) comprises an array of nucleic acid capture probes (130), the identifier is a portion of the array, such as a nucleic acid capture probe (130) or a colored molecule, fluorescent molecule in the array Alternatively, it can contain photoelectrochemical molecules. If the identifier is a nucleic acid capture probe (130), the identifier target nucleic acid (140) is added to the identifier nucleic acid capture probe (130) and hybridized, thereby forming a nucleic acid complex that is identified. Irradiating this complex with a light source (170) during the intercalation of the discriminating nucleic acid complex and the threaded PIND-Ru-PIND bis-intercalator (150) generates a photoelectrochemical signal Produce. In another embodiment, the support (110) includes means for generating an electrical signal upon irradiation of the light source (170), such as photovoltaic means, photoresist means or other photosensitive means, thereby irradiating. Identify the location of.

  The working electrode (120) is any electrode known in the art that is compatible with the disclosed assay and comprises at least one of diamond, glassy carbon, gold, graphite, indium tin oxide, platinum or silicon. Can be included. Preferably, the working electrode (120) comprises an indium tin oxide electrode. In some embodiments, the optical arrangement of these components is performed using any means known in the art, for example, by printing, coating, or either physical vapor deposition or chemical vapor deposition. ) A working electrode (120) arranged as a top layer can be involved. The working electrode (120) has a suitable physical shape, for example rectangular, circular or any polygon. Further, the working electrode (120) can be either planar or non-planar. Non-planar examples include any prism such as a cylinder, cone, or pyramid.

Nucleic acid capture probes (130) include, but are not limited to, for example, DNA, cDNA, gDNA, RNA, cRNA, tRNA, mRNA, rRNA, RNAi, iRNA, shRNA, PNA or LNA, or any combination thereof It can include any form of nucleic acid that is not. The nucleic acid capture probe (130) was designed to be a sequence that is complementary to the sequence of the target nucleic acid (140) so as to provide hybridization or annealing of the nucleic acid capture probe (130) and the target nucleic acid (140). It can be any sequence. In some embodiments, the control nucleic acid capture probe (130) can be a sequence that differs by at least one nucleotide residue from the sequence of the target nucleic acid (140). The nucleic acid capture probe (130) is coupled to the working electrode (120). In some embodiments, the working electrode (130) is composed of indium tin oxide (ITO) and pretreated by silanization, and the nucleic acid capture probe (130) is immobilized on the working electrode (120). Is an aldehyde modified. Preferably, an aliquot of denatured aldehyde-modified nucleic acid capture probe (130) is dispensed onto the silanized working electrode (120) and incubated at a temperature of 20 ° C. for a period of 2-3 hours. Thereafter, the working electrode (120) is immersed in a vigorously stirred hot water at a temperature ranging from 90 ° C. to 95 ° C. for 2 minutes. The working electrode (120) coated with the nucleic acid capture probe (130) is further impregnated in an ethanolic solution for a period of up to 24 hours. Preferably this impregnation period is in the range of 1 to 7 hours. Even more preferably, the impregnation period is in the range of 3-5 hours. The surface density of the nucleic acid capture probe (130) immobilized on the working electrode (120) can range from 5 to 8 × 10 ⁻¹² mol / cm ² . In another embodiment, the nucleic acid capture probe (130) binds to the working electrode (120) using a binding group, the identity of which will depend on the composition of the working electrode (120). For example, binding of a nucleic acid capture probe (130) to a gold electrode by use of a thiol binding group is well known in the art, and binding of a nucleic acid capture probe (130) to a diamond electrode by use of a diazonium binding group is The attachment of nucleic acid capture probe (130) to a silicon electrode by use of a substituted alkoxysilane linking group, well known in the art, is well known in the art, and nucleic acid capture probe (130) by use of an amine or carbodiimide linking group ( Binding of 130) to a glassy carbon electrode is well known in the art, and binding of a nucleic acid capture probe (130) to another electrode by use of a carboxylate-amine functional group and a biotin-avidin bond is known in the art. Well known in the field.

  Nucleic acid capture probes (130) include, but are not limited to, for example, DNA, cDNA, gDNA, RNA, cRNA, tRNA, mRNA, rRNA, RNAi, iRNA, shRNA, PNA or LNA, or any combination thereof It is designed to hybridize to a target nucleic acid (140) of any sequence, not any form. A hybridization event can result in the formation of DNA-DNA homoduplex, RNA-RNA homoduplex or DNA-RNA homoduplex or any other nucleic acid complex. The hybridization efficiency of the target nucleic acid (140) and the nucleic acid capture probe (130) is not limited to 10% to 75%, 20% to 40%, 25% to 35%, 27% to 35%, 30% to It may be within a range including 35%, 31% to 35%, 31% to 34%, 31% to 33% or 30% to 33%. Preferably, the hybridization efficiency is about 32%, indicating that 10% of the target nucleic acid (140) hybridizes to the nucleic acid capture probe (130).

The stitched PIND-Ru-PIND bis-intercalator (150) is a photoelectrochemical intercalator that binds preferentially to double-stranded nucleic acids rather than single-stranded nucleic acids. In some embodiments, the intercalation of the double-stranded nucleic acid complex and the stitched PIND-Ru-PIND bis-intercalator (150) is derived from the stitched PIND-Ru-PIND bis-intercalator (150). A stable adduct is formed by intercalation of the two naphthalene diimide groups and double-stranded nucleic acid complex, which is composed of the phosphate group of the double-stranded nucleic acid complex and the stitched PIND-Ru-PIND. An ion pair is formed with the divalent cationic Ru (bpy) ₂ ²⁺ group of the bis-intercalator (150).

  The application of voltage to the interstitial threaded PIND-Ru-PIND bis-intercalator (150) and stable adducts including double stranded nucleic acid complexes can be performed in the 3rd and 5th cycles, 3rd and 10th cycles. Steady state cyclic voltammograms can be provided during the first, third and twentieth cycles, between the third and fifty cycles, or after the third cycle. Steady state voltammograms include, but are not limited to, 0-1.0V, 0.1-1.0V, 0.2-1.0V, 0.3-1.0V, 0.4-1.0V, 0.5-1.0V, 0.5-0.9V, 0.5-0.95V, When applying voltage to Ag / AgCl electrode in the range including 0.55-0.9V, 0.6-0.9V, 0.6-0.95V, 0.65-0.9V, 0.65-0.95V or 0.6-1.0V, the potential scanning speed is 10OmV Can be realized in seconds. This steady state voltammogram shows the high stability of the threaded PIND-Ru-PIND bis-intercalator (150) when intervening in a double-stranded nucleic acid complex.

  Evaluation of the intervening stitched PIND-Ru-PIND bis-intercalator (150) as a potential redox activity indicator has a detection limit of 0.50 nM, and without limitation 0.5-550 nM, 0.5-150 nM, 0.5 Operating range including ~ 100nM, 0.5 ~ 75nM, 0.5 ~ 50nM, 0.5 ~ 20nM, 0.5 ~ 5nM, 0.8 ~ 5nM, 0.8 ~ 10nM, 0.8 ~ 15nM, 0.8 ~ 25nM, 0.8 ~ 50nM, 0.8 ~ 100nM or 0.8 ~ 200nM Can be provided. The number of intervening threaded PIND-Ru-PIND bis-intercalator (150) molecules, as determined by integration of oxidation or reduction current peaks at low scan rates, is not limited to 0.1-0.5 μC, 0.25 to 0.35 μC, 0.25 to 0.33 μC, 0.25 to 0.32 μC, 0.25 to 0.31 μC, 0.25 to 0.30 μC, 0.26 to 0.35 μC, 0.27 to 0.35 μC, 0.27 to 0.35 μC, 0.28 to 0.35 μC , 0.27 to 0.31 μC, or 0.28 to 0.30 μC. The ratio of intervening threaded PIND-Ru-PIND bis-intercalator (150) molecule to base pair of hybridized nucleic acid capture probe (130) target nucleic acid (140) complex is, without limitation, 1 / 1 to 1/20, 1/1 to 1/10, 1/2 to 1/10, 1/3 to 1/10, 1/4 to 1/10, 1/5 to 1/10, 1/3 to 1/7, 1 / 4-1 / 7, 1 / 5-1 / 7, 1 / 5-1 / 8, 1 / 4-1 / 6, 1 / 3-1 / 6 or 1 / 5-1 / It can be within the range including 6.

The stable adduct produces a photoelectrochemical response to exposure to a light source, producing a photocurrent action spectrum in the range of 400-600 nm, more preferably 490 nm. Application of up to 10 ³ irradiation cycles over a period of 60 minutes causes photocharge and discharge currents to increase substantially linearly as the number of irradiation cycles increases, with current from the maximum level to the background level The period of descent is 1.5-5.0 seconds, 1.7-3.0 seconds, 1.7-2.0 seconds, 1.75-2 seconds, 1.8-2.0 seconds, 1.85-2.0 seconds, 1.75-2.1 seconds, 1.75-2.2 seconds 1.75-2.3 seconds, 1.75-2.4 seconds, 1.75-2.5 seconds, 1.75-3.0 seconds, 1.8-2.1 seconds, 1.8-2.2 seconds, 1.8-2.3 seconds, 1.8-2.4 seconds, 1.8-2.5 seconds, 1.8-3.0 seconds , 1.85 to 2.0 seconds, 1.85 to 2.1 seconds, 1.85 to 2.2 seconds, 1.85 to 2.3 seconds, 1.85 to 2.4 seconds, 1.9 to 3.0 seconds, 1.9 to 2.5 seconds. Intervention the threading PIND-Ru-PIND bis - stability and sensitivity of intercalator (150) include, but are not limited to ^{1.0M~1 × 10 -17 M, 1.0M~1 ×} 10 -16 M, 1.0M -1x10 ^-15 M, 1x10 ^-3 M to 1x10 ^-17 M, 1x10 ^-3 M to 1x10 ^-16 M, 1x10 ^-3 M to 1x10 ^-15 M, 1 x 10 ^-6 M to 1 x 10 ^-17 M, 1 x 10 ^-6 M to 1 x 10 ^-16 M, 1 x 10 ^-6 M to 1 x 10 ^-15 M, 1 x 10 ^-9 M to 1 × 10 ^-17 M, 1 × 10 ^-9 M to 1 × 10 ^-16 M, 1 × 10 ^-9 M to 1 × 10 ^-15 M, 1 × 10 ^-12 M to 1 × 10 ^-17 M, 1 × 10 ^-12 M to 1 x 10 ^-16 M, 1 x 10 ^-12 M to 1 x 10 ^-15 M, 1 x 10 ^-13 M to 1 x 10 ^-17 M, 1 x 10 ^-13 M to 1 x 10 ^{-16 M, 1 × 10 -13 M~1} × 10 -15 M, 1 × 10 -14 M~1 × 10 -17 M, 1 × 10 -14 M~1 × 10 -16 M, 1 × 10 - ¹⁴ M to 1 x 10 ^-15 M, 1 x 10 ^-10 M to 1 x 10 ^-16 M, 1 x 10 ^-11 M to 1 x 10 ^-16 M, 1 x 10 ^-13 M to 1 x 10 ^-16 M, 1 × 10 ⁻¹⁴ M to 1 × 10 ⁻¹⁶ M, 1 × 10 ⁻¹⁵ M to 1 × 10 ⁻¹⁶ M, or 1 × 10 ⁻¹⁵ M to 5 × 10 ⁻¹⁷ M Detection of the target nucleic acid (140) can be provided. Dilution and / or titration of the target nucleic acid (140) may be required when using a concentration that can saturate the nucleic acid capture probe (130).

The photocharge / discharge current can increase linearly with incident light intensity, which includes, but is not limited to, 0.05 mW / cm ^{2 to} 20.0 mW / cm ² , 0.05 mW / cm ^{2 to} 19.9 mW / cm ^{2 , 0.05mW / cm 2 ~19.8mW / cm} 2, 0.05mW / cm 2 ~19.7mW / cm 2, 0.05mW / cm 2 ~19.6mW / cm 2, 0.05mW / cm 2 ~19.5mW / cm 2, 0.05 ^{mW / cm 2 ~19.4mW / cm 2} , 0.05mW / cm 2 ~19.3mW / cm 2, 0.05mW / cm 2 ~19.2mW / cm 2, 0.05mW / cm 2 ~19.1mW / cm 2, 0.05mW / ^{cm 2 ~19.0mW / cm 2, 1.0mW} / cm 2 ~20.0mW / cm 2, 1.0mW / cm 2 ~19.9mW / cm 2, l.0mW / cm 2 ~19.8mW / cm 2, 1.0mW / cm ^{2 ~19.7mW / cm 2 1.0mW / cm} 2 ~19.6mW / cm 2, 1.0mW / cm 2 ~19.5mW / cm 2, 1.0mW / cm 2 ~19.4mW / cm 2, 1.0mW / cm 2 ~19.3 ^{mW / cm 2 1.0mW / cm 2} ~19.2mW / cm 2, 1.0mW / cm 2 ~19.1mW / cm 2, 1.0mW / cm 2 ~19.0mW / cm 2, 2.5mW / cm 2 ~20.0mW / cm ² , 2.5 mW / cm ^{2 to} 19.9 mW / cm ² , 2.5 mW / cm ^{2 to} 19.8 mW / cm ² , 2.5 mW / cm ^{2 to} 19.7 mW / cm ² , 2.5 mW / cm ^{2 to} 19.6 mW / cm ² , ^{2.5mW / cm 2 ~19.5mW / cm 2} , 2.5mW / cm 2 ~19.4mW / cm 2, 2.5mW / cm 2 ~19.3mW / cm 2, 2.5m ^{W / cm 2 ~19.2mW / cm 2} , 2.5mW / cm 2 ~19.1mW / cm 2, 2.5mW / cm 2 ~19.0mW / cm 2, 2.6mW / cm 2 ~20.0mW / cm 2, 2.6mW / ^{cm 2 ~19.9mW / cm 2, 2.6mW} / cm 2 ~19.8mW / cm 2, 2.6mW / cm 2 ~19.7mW / cm 2, 2.6mW / cm 2 ~19.6mW / cm 2, 2.6mW / cm 2 ^{~19.5mW / cm 2, 2.6mW / cm} 2 ~19.4mW / cm 2, 2.6mW / cm 2 ~19.3mW / cm 2, 2.6mW / cm 2 ~19.2mW / cm 2, 2.6mW / cm 2 ~19.1 mW / cm ^z , 2.6 mW / cm ^{2 to} 19.0 mW / cm ² , 2.7 mW / cm ^{2 to} 20.0 mW / cm ² , 2.7 mW / cm ^{2 to} 19.9 mW / cm ² , 2.7 mW / cm ^{2 to} 19.8 mW / ^{cm 2, 2.7mW / cm 2 ~19.7mW} / cm 2, 2.7mW / cm 2 ~19.6mW / cm 2, 2.7mW / cm 2 ~19.5mW / cm 2, 2.7mW / cm 2 ~19.4mW / cm 2 ^{, 2.7mW / cm 2 ~19.3mW / cm} 2, 2.7mW / cm 2 ~19.2mW / cm 2, a 2.7mW / cm ² ~19.1mW / cm ² or ^{2.7mW / cm 2 ~19.0mW / cm 2} , It is a range to include.

  The threaded PIND-Ru-PIND bis-intercalator (150) is in the “excited” state and, when it is intervening in a double-stranded nucleic acid complex, it is “intervening” It is believed that it is more easily oxidized than in the state. Therefore, the sewn PIND-Ru-PIND bis-intercalator (150) of the working electrode (120) in the excited (interventional) state, not in the basal (non-intercalated) state, Bias to a potential sufficient to oxidize) results in oxidation of only the intervening threaded PIND-Ru-PIND bis-intercalator (150). In some embodiments, the oxidized intervening stitched PIND-Ru-PIND bis-intercalator (150) continues to bind to the double stranded nucleic acid complex. In another embodiment, the oxidized intervening stitched PIND-Ru-PIND bis-intercalator (150) is reduced to the ground state by a sacrificial reductant, whereby the sacrificial reducing agent is used. It causes a photocurrent until either it is fulfilled or the irradiation is interrupted.

  The data capture device (160) includes, but is not limited to, acting or controlling a potentiostat to control or measure voltage and / or current in the system (100), Control the system (100). The potentiostat can be any electronic device that controls the voltage difference between the working electrode (120) and the reference electrode (190). The potentiostat can perform this control by injecting current through the counter electrode (180) into the system (100). The potentiostat can measure the current flow between the working electrode (120) and the other electrode. The controlled variable of the potentiostat may be the system potential and the measured variable may be the system current. This potentiostat allows the control voltage to force the current through a counter electrode (180) that is high enough to achieve the desired potential difference between the working electrode (120) and the reference electrode (190). Can cause. The control voltage can be generated by an internal potential control power source of the potentiostat or by an external signal generator, such as a ramp generator or a sine generator. The potentiostat can be used as a controlled and precise voltage source so that the potential supplied to the system (100) (or set by an internal voltage source) directly causes the voltage at the counter electrode (180). Can be controlled. The maximum current may be limited by the set current range and by the power of the potentiostat, so that a voltage applied beyond the control voltage range requires the insertion of a potentiometric voltage divider, thereby causing a potentiostat Increase the voltage amplification. Alternatively, the potentiostat can function as a controlled current source or as a precision ammeter. In addition, the potentiostat may be capable of measuring currents as small as single electrons using, for example, quantum dots, scanning probe devices, and single electron tunnel devices. The photocurrent can be measured using a fluorescence spectrophotometer.

  In some embodiments, the data capture device (160) modulates a light source (170) that includes, but is not limited to, one or more portions of the illuminated working electrode (120). For example, the data capture device (160) can go to the light source (170) and scan the working electrode (120) in a predetermined pattern. This is useful in embodiments where the working electrode (120) includes an array of nucleotide capture probes (130) linked to the working electrode (120). In another embodiment, the data capture device (160) optionally receives data from an optical scanner or detector (220) configured to receive optical information from either the working electrode (120) or the support (110). receive. For example, the optical information can include an optically encoded sample identifier, such as a barcode. Alternatively, the data capture device (160) is either from a working electrode (120) or support (110) encoded by other means, for example by using other integrated devices such as radio frequency tags or microprocessors. Configured to receive sample identification data. In a further embodiment, the data capture device (160) implements certain operating procedures in the system (100) upon receiving specific encoded sample identification data. In yet another embodiment, the optical scanner or detector (220) comprises a working electrode (120) generated by a photoelectrochemical reaction elicited from an intervening threaded PIND-Ru-PIND bis-intercalator (150). , And then such fluorescence data is sent to a data capture device (160). In another embodiment, the data capture device (160) includes, but is not limited to, a counter electrode (180), a reference electrode (190), a fluid system (200), a temperature controller (210), and / or an optical scanner or detector. Data from optical components, including (220), can be acquired, processed and reacted. Although this data capture device (160) is illustrated as a single component, it may include several components. These components can be specific to a particular function of this system (100) and can include modular components, thereby allowing specific configurations depending on the desired function of the system (100). Give users the flexibility to add or remove parts. The data capture device 160 may include at least one interface for data acquisition known in the art, such as a display, keyboard or keypad, printer, and / or peripheral data port. The data capture device (160) may be programmable by the user or pre-programmed, or a combination of both.

  The light source (170) includes any source of sufficient intensity and energy to be able to induce a photoelectrochemical reaction from the threaded PIND-Ru-PIND bis-intercalator (150). Suitable light sources are lasers, arc lamps, light emitting diodes (LEDs) such as blue or blue light emitting diodes, fluorescent lamps, halogen lamps, metal halide lamps, discharge lamps such as xenon discharge lamps, tungsten incandescent lamps, high pressure sodium Including but not limited to lamps or sunlight. The selected light source is at least near 490 nm or 490 nm light, e.g. about 400-600 nm, 410-590 nm, 420-580 nm, 430-570 nm, 440-560 nm, 450-550 nm, 460-540 nm, 450-550 nm, 460-540nm, 470-530nm, 480-520nm, 480-510nm, 480-500nm, 481-499nm, 482-498nm, 483-497nm, 484-496nm, 485-495nm, 486-494nm, 487-493nm, 488- It emits light in the range of 492 nm or 489 to 491 nm. This light can be infrared, visible or ultraviolet. In some embodiments, the light source (170) is configured to direct electromagnetic radiation to the working electrode (120) or a portion thereof. In another embodiment, the light source (170) is configured to scan the surface of the working electrode (120) in a predetermined pattern. In some embodiments, the light source (170) scans the working electrode (120) by moving or by moving the working electrode (120). A laser is a particularly useful light source (170) for a system (100) that includes an array of nucleic acid capture probes (130) coupled to a working electrode (120).

  Optional counter electrode (180) and reference electrode (190) are of any type known in the art suitable for performing the present invention. Preferably, the counter electrode (180) is a platinum wire and the reference electrode (190) is a silver / silver chloride electrode.

  Any fluid system (200) is suitable in the art suitable for carrying out the present invention, for example for dispensing a sample onto the working electrode (120), washing the working electrode (120), or adding reagents. Any known type. The fluid system (200) can be provided for continuous flow-through control of fluid at a predetermined rate.

  The optional temperature control device (210) can, for example, support (110), working electrode (120), and / or fluid in contact with support (110) and / or working electrode (120) as needed. Any type known in the art suitable for carrying out the present invention for heating or cooling.

  The optional optical scanner or detector (220) is any type known in the art suitable for carrying out the present invention. The optical scanner or detector (220) can be an ultraviolet / visible / near infrared spectrometer.

  The present invention thus provides an electrode system for detecting and / or quantifying a target nucleic acid. The nucleic acid capture probe (130) linked to the working electrode (120) is hybridized with the target nucleic acid (140) to form a nucleic acid complex, which then contains a threaded PIND-Ru-PIND. When the bis-intercalator (150) intervenes and irradiation with a fixed wavelength light source results in the generation of a photoelectrochemical reaction from the intervening threaded PIND-Ru-PIND bis-intercalator (150), A photocurrent action spectrum is produced. The application of the irradiation cycle results in a photocharge and discharge current proportional to the amount of intervening stitched PIND-Ru-PIND bis-intercalator (150). This current measurement therefore provides a measure of the amount of intervening stitched PIND-Ru-PIND bis-intercalator (15O), with stitched PIND-Ru-PIND bis-intercalator (150) preferentially. This will then be a measure of the amount of hybridized target nucleic acid (140) as it intervenes in the double stranded nucleic acid complex.

The present invention also provides biosensor “chips”, otherwise known as arrays or microarrays. The biosensor chip comprises a nucleic acid capture probe (130) comprising a chip, at least one working electrode (120) disposed on the chip, a sequence coupled to the working electrode (120) and complementary to the sequence of the target nucleic acid (140). ), As well as a threaded PIND-Ru-PIND screw-intercalator (150). Thus, in some embodiments, the working electrode (120) disposed on the support (110) comprises an array, microarray or “chip” having a plurality of nucleic acid capture probes (130) linked thereto. The entire array may be disposed on a single working electrode (120) or across a plurality of working electrodes (120) each disposed on a support (110). In another embodiment, the array of nucleic acid capture probes (130) is disposed on one or more working electrodes (120) that are bound to either one or more surfaces of the support (110). Is done. The array is placed on the working electrode (120) by any suitable method known in the art, such as pipette, ink jet printing, contact printing or photolithography. The array is composed of at least one element, each element including at least one nucleic acid capture probe (130). At least one element may be composed of multiple nucleic acid capture probes (130) of the same sequence. The number of elements constituting the array can be any number from 1 to 10 ⁹ or more. When multiple elements are arranged on an array, the array elements can be spaced at a uniform distance or a variable distance, or a combination thereof. The distance between the centers of each array element may be a distance suitable for performing the present invention, such as 100 μm, 10 μm, 1 μm, or any other distance. In some embodiments, the array elements are randomly placed, after which each position of each array element is determined. The size and shape of the array elements depends on the particular application of the invention, and elements of different sizes and shapes can be combined in a single array. The surface of the array may be substantially planar or may have features such as a recess or ridge, and the array elements can be placed either in the recess or on the ridge. Such a recess can provide a reservoir of solution into which the array element is impregnated, or such a ridge facilitates drying of the array element, as is necessary for the performance of the present invention. be able to. For example, elements may be placed in each well of a 96 well plate. In some embodiments, the working electrode (120) and / or the support (110) is used to identify each of the array elements, such as indicia, radio frequency tags, integrated devices such as microprocessors, barcodes or other indicia. A unique identifier such as The unique identifier can additionally or alternatively include a recess or ridge on the surface of the array. Furthermore, a unique identifier can be provided for precise orientation or identification of the working electrode (120) on which the array is disposed. The unique identifier can be read directly by the data capture device (160) or by the optical scanner or detector (220). In use, the biosensor chip may include an array of nucleic acid capture probes (130) having a sequence complementary to the sequence of the target nucleic acid (140) and linked to the working electrode (120). Involved in the target nucleic acid (140) that hybridizes with the nucleic acid capture probe (130) to form a strand nucleic acid complex, which in turn contains a threaded PIND-Ru-PIND bis-intercalator (150 The amount of PIND-Ru-PIND (150) intervening indicates the amount of target nucleic acid.

  The present invention comprises a step of hybridizing a target nucleic acid (140) to a nucleic acid capture probe (130) linked to a working electrode (120) disposed on a support (110), whereby a double-stranded nucleic acid complex And then interposing a threaded PIND-Ru-PIND bis-intercalator (150) into the double-stranded nucleic acid complex, and then illuminating the PIND-Ru-PIND bis-intercalator (150). Irradiating the working electrode (120) and / or the support (110) with light containing at least one wavelength to be activated, then applying a potential to the working electrode (120), and then applying a photocurrent action spectrum. There is also provided a method for detecting and / or quantifying a target nucleic acid, comprising the step of obtaining, wherein the amount of target nucleic acid (140) is determined from a photocurrent action spectrum.

  The step of hybridizing the target nucleic acid (140) to the nucleic acid capture probe (130) depends on the complementarity between each sequence of the target nucleic acid (140) and the nucleic acid capture probe (130). If each of these sequences are complementary, the target nucleic acid (140) will hybridize to the nucleic acid capture probe (130). If each sequence is not complementary, the target nucleic acid (140) will not effectively hybridize to the nucleic acid capture probe (130) in a manner suitable for carrying out the present invention. The target nucleic acid (140) can be dissolved in a suitable solvent, such as water, organic solvent or aqueous buffer, or combinations thereof. In some embodiments, the target nucleic acid (140) is hybridized with the nucleic acid probe (130) by the fluid system (200) controlled in some embodiments by the data capture device (160). In another embodiment, the temperature controller (210) can be used to vary the temperature at which the hybridization step occurs. The temperature controller (210) can be controlled by the data capture device (160). The conditions or stringency selected upon hybridization of the target nucleic acid (140) to the nucleic acid capture probe (130) include, but are not limited to, the type of nucleic acid involved, the target nucleic acid (140) and the nucleic acid capture probe (130). It will vary depending on various parameters, including the sequence and the solvent in which the target nucleic acid (140) is dissolved. Such conditions are known to those skilled in the art. For example, if either the nucleic acid capture probe (130) or the target nucleic acid (140) is double stranded, this double stranded form is denatured into a single stranded form prior to hybridization. Optimal hybridization conditions can be determined by the melting temperature (Tm) of both the target nucleic acid (140) and nucleic acid capture probe (130), which is present in the target nucleic acid (140) and nucleic acid capture probe (130). It can be calculated by assigning a value of 2 ° C. to each A or T residue and assigning a value of 4 ° C. to each G or C residue. The rate of hybridization can be optimal when performed using temperatures below about 20-30 ° C. below the Tm temperature, but this can vary significantly depending on the particular application of the method. The present invention can be suitably performed by hybridization of the target nucleic acid (140) to all or part of the nucleotide residues including the nucleic acid capture probe (130), so that the target nucleic acid (140) and / or Either one or both of the nucleic acid capture probes (130) may have a single stranded portion after the hybridization step.

  Optionally, after the hybridization step, the support (110) having a working electrode (120) disposed on the support and a linked nucleic acid capture probe (130) hybridized to the target nucleic acid (140) is Washing is performed to remove target nucleic acid (140) that has not completely hybridized to probe (130). Washing effectively removes incompletely hybridized target nucleic acid (140) but retains fully hybridized target nucleic acid (140) to nucleic acid capture probe (130) and is known to those skilled in the art. It can be realized by any method. This cleaning step can involve the regulation of the fluid system (200) and / or the temperature controller (210), either or both of which may be dependent on the stringency of the cleaning step as needed for the particular application of the method. Can be controlled by the data capture device (160). Stringency can also vary with the composition of the cleaning fluid. Optimization of wash stringency can be achieved by methods known to those skilled in the art and can include, for example, nucleic acid length and composition calculations, wash temperature, and salt concentration.

Thereafter, an embedded PIND-Ru-PIND bis-intercalator (150) intervenes in the double-stranded nucleic acid complex formed by hybridization of the target nucleic acid (140) and the nucleic acid capture probe (130). In some embodiments, the intercalation process is realized through the use of a fluid system (200) that can be controlled by the data capture device (160). In another embodiment, a plurality of stitched PIND-Ru-PIND bis-intercalators (150) are used. In yet another embodiment, both the target nucleic acid (140) and the threaded PIND-Ru-PIND bis-intercalator (150) are applied to the nucleic acid capture probe (130) simultaneously. The stitched PIND-Ru-PIND bis-intercalator (150) binds preferentially to double-stranded nucleic acid complexes but not to single-stranded nucleic acids. Therefore, the threaded PIND-Ru-PIND bis-intercalator (150) allows the target nucleic acid (140) to hybridize to the nucleic acid capture probe (13O) and to capture single-stranded target nucleic acid (140) or single-stranded nucleic acid. It binds only to nucleic acid regions that do not bind to probe (130). In one preferred embodiment, the intercalation of the double-stranded nucleic acid complex and the threaded PIND-Ru-PIND bis-intercalator (150) is derived from the threaded PIND-Ru-PIND bis-intercalator (150). A stable adduct is formed by the intercalation of two naphthalene diimide groups and double-stranded nucleic acid complex, and the phosphate group of the double-stranded nucleic acid complex and the stitched PIND-Ru-PIND bis-inter An ion pair is formed with the dicationic Ru (bpy) ₂ ²⁺ group of the calator (150). Optimization of the intercalation of the threaded PIND-Ru-PIND bis-intercalator (150) includes, but is not limited to, the length of hybridization between the target nucleic acid (140) and the nucleic acid capture probe (130), As well as factors including the concentration of the target nucleic acid (140), nucleic acid capture probe (130) and threaded PIND-Ru-PIND bis-intercalator (150). Each of these and possible other factors can be optimized by one skilled in the art.

  After intercalation of the threaded PIND-Ru-PIND bis-intercalator (150) and the double-stranded nucleic acid complex, the working electrode (120) is driven by a light source (170) controlled by a data capture device (160). Irradiated. This irradiation mainly excites only the intervening stitched PIND-Ru-PIND bis-intercalator (150) and not the "basal" non-interventional stitched PIND-Ru-PIND bis-intercalator (150). May be calibrated. Irradiation optimization is a factor that includes, but is not limited to, the excitation efficiency of the stitched PIND-Ru-PIND bis-intercalator (150) during intercalation and its concentration if a sacrificial reducing agent is used It is thought that it depends on. In some embodiments, the light source (170) intervened a threaded PIND-Ru-PIND bis-intercalator (150) by either movement of the light source (170) or movement of the working electrode (120). It is possible to scan an array of hybridized nucleic acid complexes. In another embodiment in which an array exists, the light source can scan individual array elements. The potential of the working electrode (120) is controlled by the data capture device (160), which oxidizes the excited intervening threaded PIND-Ru-PIND bis-intercalator (150) but not the basal non- The intervention threaded PIND-Ru-PIND bis-intercalator (150) can be biased to a potential that cannot be oxidized. In some embodiments, the nucleic acid capture probe (130) is optionally contacted with a sacrificial reducing agent, which oxidizes the intervening threaded PIND-Ru-PIND bis-intercalator (150) from an excited state. It can be reduced to the ground state. This allows the stitched PIND-Ru-PIND bis-intercalator (150) to generate a continuous photocurrent during the irradiation period, which can be measured by the data capture device (160). In certain embodiments, the working electrode (120) does not directly oxidize the sacrificial reducing agent, thus minimizing the possibility of interfering with background current. The composition of the sacrificial reducing agent is any known to those skilled in the art capable of performing the present invention, such as tertiary amine, ethylenediaminetetraacetic acid, tripropylamine and β-lactam.

  The process of obtaining the photocurrent action spectrum of the working electrode is related to the measurement of the current at the working electrode (120) by any means known to those skilled in the art suitable for carrying out the present invention. In one embodiment, the current is measured by a data capture device (160).

  The present invention also provides an apparatus for detecting and / or quantifying a target nucleic acid. This device comprises an electrode system (100), a light source (170) as a means for irradiating the working electrode (120) of the electrode system (100), and a means for applying a potential to the working electrode (120). A data capture device (160) and a data capture device (160) as a means for obtaining a photocurrent action spectrum can be included. The apparatus optionally further comprises a data capture device (160), a fluid system (200), a temperature controller (210) and an optical or detector (220) for analyzing the photocurrent action spectrum. Irradiation of the working electrode (120) of the electrode system (100) by means for irradiating the working electrode (120) of the electrode system (100) and working electrode by means for applying a potential to the working electrode (120) Application of a potential to 120) results in a photocurrent action spectrum obtained by means for obtaining a photocurrent action spectrum. The amount of target nucleic acid is then determined from the photocurrent action spectrum.

  Further improvements in the sensitivity and specificity of this method are further minimizing background caused by more selective photoreporter design, optimization of hybridization conditions, and uptake independent of intercalator hybridization. It is planned through conversion.

  The present invention will now be further described in more detail with reference to the following specific examples, which should not be construed as limiting the scope of the invention in any way.

2. 合成核酸の捕獲プローブ：

3. 対照バイオセンサーの捕獲プローブ：

4. TP53の捕獲プローブ1：

5. TP53の捕獲プローブ2：

6. 単ミスマッチ塩基による捕獲プローブ1：

7. 単ミスマッチ塩基による捕獲プローブ2：

8. 単ミスマッチ塩基による捕獲プローブ3：

9. 単ミスマッチ塩基による捕獲プローブ4：

10. 単ミスマッチ塩基による捕獲プローブ5：

11. 単ミスマッチ塩基による捕獲プローブ6：

A₆は、ポリアデニル化配列AAAAAAを意味する。 Examples Example 1. General Methods and Materials Oligonucleotides The oligonucleotide sequences used to illustrate the present invention are as follows:
1. Synthetic target nucleic acid:

2. Synthetic nucleic acid capture probes:

3. Control biosensor capture probe:

4. TP53 capture probe 1:

5. TP53 capture probe 2:

6. Capture probe with single mismatch base 1:

7. Single probe mismatch probe 2:

8. Single mismatch base capture probe 3:

9. Single mismatch base capture probe 4:

10. Single probe mismatch probe 5:

11. Single probe mismatch probe 6:

A ₆ refers to the polyadenylation sequence AAAAAA.

Light reporter
PIND-Ru-PIND was prepared from [Ru (bpy) ₂ ] Cl ₂ and PIND as previously described ¹⁰ .

Apparatus Electrochemical experiments were performed using a CH Instruments model 660A electrochemical workstation (CH Instruments, Austin, TX). A conventional electrode consisting of an indium tin oxide (ITO) working electrode (0.20 ± 0.02 cm ² ), a nonleak Ag / AgCl (3.0 M NaCl) reference electrode (Cypress Systems, Lawrence, KS), and a platinum wire counter electrode A 3-electrode system was used for all electrochemical measurements. Ultraviolet (UV) visible spectra were recorded on a V-570 UV / visible (VIS) / near infrared (NIR) spectrophotometer (JASCO Corp., Japan). Photocurrent measurements were performed with a Fluorolog®-3 fluorescence spectrophotometer (Jobin Yvon Inc, Edison, NJ) in combination with a synchronized 660A electrochemical workstation. The display mode of the fluorescence spectrophotometer was adapted to photocurrent action experiments at 10 nm intervals. The intensity of monochromatic light incidence on the ITO electrode was controlled by adjusting both the slit width and the distance to the illuminator. Light intensity at 490 nm was calibrated with a Newport model 841-PE energy and power meter (Newport Corp., Irvine, Calif.). Irradiation was performed from the front side of the ITO electrode to prevent light absorption by the glass substrate. The three electrodes were hosted in a standard 1.0 cm fluorescent cuvette, with the working electrode facing the irradiation window and the other two electrodes placed behind the working electrode. All potentials reported in this work relate to Ag / AgCl electrodes. All experiments were performed at room temperature unless otherwise noted.

Biosensor preparation, hybridization and detection
Pretreatment and silanized ITO electrode were carried out according to the method ³² of Russell et al. Immobilization of the oligonucleotide capture probe was performed as follows: the aldehyde-modified capture probe was denatured at 90 ° C. for 10 minutes to a concentration of 0.50 μM in 0.10 M acetate buffer (pH 6.0). Diluted. A 25 μl aliquot of the capture probe solution was dispensed onto the silanized electrode and incubated for 2-3 hours at 20 ° C. in an environmental chamber. After incubation, the electrode was rinsed successively with 0.10% sodium dodecyl sulfate (SDS) and water. The imine reduction was performed by incubation of the electrode for 5 minutes in a 2.5 mg / ml sodium borohydride solution made with phosphate buffered saline (PBS) / ethanol (3/1). The electrode was then immersed in hot water (90-95 ° C.) with vigorous stirring for 2 minutes, rinsed with plenty of water, and air dried with a stream of nitrogen. In order to minimize PIND-Ru-PIND uptake independent of hybridization and to improve the quality and safety of the electrode coated with the capture probe, the electrode coated with this capture probe was treated with 11-aminoundecanoic acid ( AUA) in 2.0 mg / ml ethanolic solution for 3-5 hours. Unreacted AUA molecules were rinsed away, and the electrode was washed by impregnation in stirred ethanol for 10 minutes, followed by a complete rinse with ethanol and water. The surface density of the immobilized capture probe, electrochemically evaluated using Tarlov's method ¹³ , is found to be in the range of 5.0 to 8.0 × 10 ^-12 mol / cm ² , which is gold This is probably 20-25% lower than the values observed for the electrodes, probably due to the lower capture probe immobilization efficiency due to chemical coupling. Hybridization of the target nucleic acid and its photoelectrochemical detection were performed in three steps. First, the biosensor was placed in a ^zero environmental chamber maintained at 5O ° C. A 25 μl aliquot of hybridization solution containing the target nucleic acid was evenly seeded on the biosensor. This was then hybridized for 60 minutes and then rinsed thoroughly with a blank hybridization solution at 50 ° C. After incubation for 20 minutes at 25 ° C. with a 25 μl aliquot of 50-100 μM PIND-Ru-PIND in Tris-ethylenediaminetetraacetic acid (TE) buffer (pH 6.0, adjusted with 10 mM HCl), PIND-Ru-PIND Binding to hybridized target nucleic acid via bis-threaded intercalation. Then it was rinsed completely with pH 6.0 TE buffer. The photocurrent was measured at 0.10 V in 0.10 M NaClO ₄ .

Example 2. Validation of gene detection by PIND-Ru-PIND photoelectrochemistry The scheme for photoelectrochemical detection of nucleic acids by direct hybridization and formation of nucleic acid / photoreporter adducts is that of electrochemical detection ¹⁰ Is different but similar. A monolayer oligonucleotide capture probe was immobilized on the ITO electrode surface via a chemical bond. The electrode was then continuously exposed to the target nucleic acid solution and the intercalator solution. Upon irradiation, a photoelectrochemical reaction (photocharging current) was generated in the system, and a discharge current was subsequently generated when irradiation was extinguished. Both charging and discharging currents were directly correlated to the target nucleic acid concentration.

In the first hybridization test, a synthetic oligonucleotide was selected as the target nucleic acid (SEQ ID NO: 1). Upon hybridization at 50 ° C. for 60 minutes, the target nucleic acid selectively bound to its complementary capture probe (SEQ ID NO: 2) and began to be immobilized on the biosensor surface. Subsequently, upon subsequent incubation with 50 μM PIND-Ru-PIND solution, PIND-Ru-PIND bound to the biosensor via screw-sewn intercalation. A typical cyclic voltammogram of the biosensor after intercalation is shown in FIG. 2A. As shown in trace 1 of FIG. 2A, a fairly high peak current was observed in the first cycle anode process, most likely due to electrocatalytic oxidation ¹² of the captured nucleic acid (guanine base). This indicates that a higher number of electrons were involved in the oxidation process. The peak current dropped significantly during continuous potential cycling and the steady state voltammogram was reached after 3 cycles between 0 and 1.0 V (Figure 2A trace 2). Subsequent excessive washing and potential cycling do not result in a noticeable change, and the intercalator binds firmly to double-stranded DNA (ds-DNA) via bio-threaded intercalation on the biosensor surface I made it clear. FIG. 2A trace 3 shows a voltammogram of an electrode (control biosensor) coated with a non-complementary capture probe after the same treatment. Negligible redox activity was observed at the redox potential of PIND-Ru-PIND. These results clearly indicate that PIND-Ru-PIND interacts selectively with ds-DNA and that the resulting ds-DNA / PIND-Ru-PIND adduct has a very slow dissociation rate. It is clear. Furthermore, there is almost no PIND-Ru-PIND incorporation unrelated to hybridization due to the presence of cationic amines on the biosensor surface.

The high level of stability of the analyte complex and photoreporter adduct is due to the presence of two naphthalenediimide groups in the ds-DNA followed by the PIND-Ru-PIND dicationic Ru (bpy) ₂ ²⁺ group. This can be explained on the basis of forming ion pairs with ds-DNA phosphate groups and significantly delaying the dissociation process.

Consequently, subsequently intervened PIND-Ru-PIND was evaluated as a potential redox activity indicator for electrochemical detection of nucleic acids. A detection limit of 0.50 nM and a dynamic range of 0.8-200 nM were obtained. The estimated hybridization efficiency of ~ 32% at the top of the dynamic range using Procedure ¹³ proposed by Tarlov and his colleagues indicates that ~ 10% of the target nucleic acids hybridized correctly. Equivalent to the values found in references ^10,14,15 . The number of intervening PIND-Ru-PIND molecules was estimated from the charge under steady state voltammograms. The integration of the oxidation or reduction current peak at low scan rates ≦ 10 mV / sec resulted in a charge of 0.29 μC and thus from 3.0 pmol of active intervening PIND-Ru-PIND. This number represents that <0.1% of PIND-Ru-PIND is contained in the assayed droplet and the PIND-Ru-PIND / base pair ratio is 1/5 to 1/6 Yes.

Example 3. Photoelectrochemical characterization of nucleic acid / PIND-Ru-PIND adducts The possibility of using intervened PIND-Ru-PIND as a photoreporter for energy conversion of nucleic acid hybridization events was tested. Trace 1 in FIG. 2B shows the photoelectrochemical reaction of the hybridized and PIND-Ru-PIND treated biosensor when the illumination is turned on or off. This photocurrent generation consisted of two steps. The first spike of photocurrent (photocharging) appeared immediately after irradiation, which then decayed rapidly to the background level. It took <2.0 seconds for the photocurrent to drop to the background level. This represents the separation of photogenerated electron-hole pairs at the biosensor surface. Under a positive bias, the holes “move” to the biosensor-solution interface, while the electrons “sank” towards the substrate electrode. Rapid disintegration of the photocurrent, the electron / most of the holes, instead of capturing electrons from or electrolyte donate electrons to the electrolyte, ¹⁶ showed that accumulated at the interface.

When the light was extinguished, similar photocurrent transient behavior (photodischarge) of the cathode current was observed. The cathode current decayed over time from the initial maximum to the background level within 2.0 seconds, which was caused by photogenerated electron and hole recombination at the biosensor surface. The photocharging / discharging cycle was regenerated more than 10 ³ times within 60 minutes without a noticeable decrease in intensity. Part of the regeneration experiment (50 cycles) is shown in the inset of FIG. 2B. In contrast, the control biosensor failed to capture any target nucleic acid and therefore no photoelectrochemical activity was observed upon irradiation (FIG. 2B trace 2). Nucleic acids have been shown to act as hole-generating biopolymers ^17-19 . Two limited mechanisms have been proposed: superexchange ²⁰ and discrete hopping ²¹ . Both mechanisms require structural distortion of the nucleic acid double helix ^22,23 . The question of how charge travels through nucleic acids over long distances remains a matter of debate. ^17-26 Nevertheless, it is generally accepted that π-stacked nucleic acid systems have their unique electronic properties and are significantly different from other biopolymers such as proteins and carbohydrates. Charge generation, separation and recombination processes occur during irradiation ^17-26 . In a more recent report, a hole transport which is optically stimulated through the ds-DNA was observed ^27. Hole transport efficiency was shown to be highly dependent on DNA sequence and potential bias. If the potential bias starts to become a smaller positive, the magnitude of the photocurrent decreases rapidly approaches the background 0.0 V, leaving only the charge separation which is optically generated in the system ^27.

Example 4 Analysis of Photocurrent Action Spectrum A plot of the photocurrent action spectrum, ie the observed photoelectrochemical reaction as a function of incident light wavelength, is shown in FIG. The photocurrent action spectrum of hybridized and intervening biosensors in the 400-600 nm region (Figure 3 Trace 2) is labeled as the PIND-Ru-PIND absorption spectrum (Figure 3 Trace 3) and is highest at 490 nm. It can be seen that this is a photocurrent, suggesting that PIND-Ru-PIND is a photoactive element on the biosensor surface. This photocurrent pattern was highly reproducible for many on-off irradiation cycles. However, in the absence of intervening light reporter, the control biosensor showed little photoactivity (Figure 3 Trace 1). Faint anode photocurrent observed in the region of 400~440nm was due primarily to suppress light electrochemical activity of the substrate electrode ^28.

Example 5. Analysis of irradiation intensity Irradiation intensity had a significant effect on photocurrent. As shown in FIG. 4, the photocharge / discharge current increased linearly with incident light intensity up to 20 mW / cm ² and leveled at higher intensity. ²⁸ linear relationship between the photocurrent and the incident light intensity, light generated charge carriers, which suggested that mono Photonics (monophotonic) process.

Example 6 Analysis of Photocurrent and Applied Potential FIG. 5 shows the dependence of the photocurrent on the applied potential. The photocharging current gradually increased when the applied potential moved in the negative direction. Since ²⁷ photogenerated charge carriers to be transported at a lower to the electrode / electrolyte efficiency, increase in light charging current with a small positive bias was caused by reduction of the charge collection efficiency. The onset potential was found to be ~ 0.60V. All photogenerated charge carriers above the onset potential were collected / lost in the interfacial reaction. As the applied potential decreased, the fraction of charge carriers collected / lost decreased. This fraction is virtually independent of light intensity, implying that charge carriers are only efficient when transported to or withdrawn from the electrode with limited photocharging current. Yes.

Example 7. Analysis of photocurrent and number of irradiation cycles Considering the extremely low dissociation rate of the nucleic acid / photoreporter adduct and the highly reversible photoelectrochemical reaction, the relationship between photocurrent and number of irradiation cycles was determined. Tested. As shown in FIG. 6, both the photocharging and discharging currents increased almost linearly up to 10 ³ cycles as the number of irradiation cycles increased. Furthermore, since the random noise tends to cancel each other after integrating over a sufficiently large number of cycles, a substantial reduction in background noise was also obtained.

Example 8. Analytical application in nucleic acid assays The applicability of photoelectrochemical methods in nucleic acid assays was tested in genomic samples. Full-length TP53 mouse cDNA was used as a standard, diluted at different concentrations with pH 8.0 hybridization buffer, and then used with a complementary capture probe (SEQ ID NOs: 4-5). Sample solutions containing different concentrations of cDNA ranging from 10 fM to 10 nM were tested. For control experiments, a non-complementary capture probe (SEQ ID NO: 3) was used in the biosensor preparation. As shown in FIG. 7, 1000 cycle integration resulted in a sensitivity enhancement of 10 ⁴ times better than the voltammetry method, with an operating range of 50 fM to 1.0 nM (R ² = 0.97), a detection limit of 20 fM.

In order to further elucidate hybridization efficiency and PIND-Ru-PIND loading levels, a series of voltammetric measurements were performed on 1.0 nM TP53 mouse cDNA after hybridization and intercalation. ~ 3.0fM of TP53 hybridizes, representing ~ 0.21% of surface-bound capture probes, much lower than that for ^8,13-15 short oligonucleotides (20-50mer) reported in the prior art It was shown that there is. In addition, voltammetric experiments showed that on average ~ 20 PIND-Ru-PIND molecules intervene in hybridized TP53. Discrepancy between hybridization efficiency and PIND-Ru-PIND loading levels, suggesting that some of the PIND-Ru-PIND molecules can intervene to the secondary structure of TP53 ^{30, 31,} the more sensitive of the method Strengthen.

Example 9. Analysis of biosensor specificity The specificity of a biosensor for target gene detection was determined by exchanging a fully complementary capture probe with a one base mismatched probe (SEQ ID NO: 6-11). And 50 ng mRNA extract. Given that there are more than 30,000 genes in this mRNA pool, the actual detectable amount of TP53 was in the range of average picograms (~ sub-picomoles). As shown in FIG. 8, the current increment of biosensors coated with perfectly matched capture probes ranged from 2.2 to 2.6 nA, whereas biosensors coated with 1 base mismatched capture probes The increment is reduced by at least 60%, as low as 0.85 nA, and is easily distinguishable between perfectly matched and mismatched genes.

References

1 is a schematic diagram of one embodiment of the present invention. (A) Cyclic voltammograms of biosensor hybridized with 50 nM target nucleic acid and intervened with PIND-Ru-PIND: (1) first scan, (2) third scan, and (3) control biosensor. Potential scanning speed 100mV / sec. (B) Photoelectrochemical reaction: (1) a biosensor hybridized with 25 nM target nucleic acid and intervened by PIND-Ru-PIND; and (2) a control biosensor. Inset: Biosensor stability test. Wavelength 490nm, light intensity 22.4mW / cm ² , applied potential 0.10V. (1) Photocurrent action spectrum of a control biosensor, (2) Photocurrent action spectrum of a biosensor hybridized with 1.0 nM target nucleic acid and treated with PIND-Ru-PIND, and (3) 25 μM PIND in H ₂ O -UV-vis absorption spectrum of Ru-PIND. Irradiation was performed with monochromatic light at intervals of 10 nm. The photocurrent was collected at 0.10V. Dependence of photocurrent on incident light intensity (400-600 nm) of a biosensor hybridized with 1.0 nM nucleic acid and intervened with PIND-Ru-PIND at 0.10 V. Dependence of photocurrent on the applied potential of an electrode coated with a complementary capture probe hybridized with 1.0 nM target DNA and treated with PIND-Ru-PIND. Irradiation was performed with a 490 nm 23.1 mW / cm ² monochromatic light beam. Multi-irradiation cycle photoelectrochemical reaction of biosensor hybridized with 100 pM target nucleic acid and treated with PIND-Ru-PIND. Wavelength 490nm, light intensity 22.8mW / cm ² . Photoelectrochemical reaction of various concentrations of TP53 mouse cDNA. 1000 cycles of integration, wavelength 490nm, light intensity 22.7mW / cm ² . Inset: Photoelectrochemical reaction at the low concentration end. Biosensor photoelectrochemical after hybridization and intercalation in a 50 ng mRNA mixture using (1) and (3) complementary capture probes, and (2) single base mismatch capture probes with mouse TP53 reaction. Wavelength 490nm, light intensity 22.9mW / cm ² .

Claims (19)

(a) working electrode;
(b) a nucleic acid capture probe linked to a working electrode, comprising a sequence complementary to the sequence of the target nucleic acid; and
(c) comprising a stable intercalator that is selective for double stranded nucleic acids and is capable of generating a photoelectrochemical reaction upon irradiation and application of a potential;
The target nucleic acid hybridizes with the nucleic acid capture probe to form a double-stranded nucleic acid complex, which intercalates a stable intercalator, and the amount of stable intercalator is the amount of target nucleic acid. As shown, the electrode system.   2. The electrode system of claim 1, wherein the stable intercalator is a threaded PIND-Ru-PIND bis-intercalator. 3. The electrode system according to claim 1, wherein the target nucleic acid can be detected when the target nucleic acid is used at a concentration of up to 1 × 10 ⁻¹⁶ M.   4. The electrode system according to any one of claims 1 to 3, wherein the working electrode comprises an array of nucleic acid capture probes. (a) hybridizing a target nucleic acid with a nucleic acid capture probe linked to a working electrode to form a double-stranded nucleic acid complex;
(b) intervening in the double-stranded nucleic acid complex with a stable intercalator that is selective for double-stranded nucleic acids and is capable of generating a photoelectrochemical reaction upon application of radiation and potential;
(c) irradiating the working electrode with light containing at least one wavelength that photoactivates a stable intercalator;
(d) applying a potential to the working electrode; and
(e) obtaining a photocurrent action spectrum,
A method for detecting a target nucleic acid, wherein the amount of the target nucleic acid is determined from a photocurrent action spectrum. (a) hybridizing a target nucleic acid with a nucleic acid capture probe linked to a working electrode to form a double-stranded nucleic acid complex;
(b) intervening in the double-stranded nucleic acid complex with a stable intercalator that is selective for double-stranded nucleic acids and is capable of generating a photoelectrochemical reaction upon application of radiation and potential;
(c) irradiating the working electrode with light containing at least one wavelength that photoactivates a stable intercalator;
(d) applying a potential to the working electrode; and
(e) obtaining a photocurrent action spectrum,
A method for quantifying a target nucleic acid, wherein the amount of the target nucleic acid is determined from a photocurrent action spectrum.   The method according to claim 5 or 6, wherein the stable intercalator is a stitched PIND-Ru-PIND bis-intercalator. The method according to any one of claims 5 to 7, wherein the target nucleic acid can be detected and quantified when the target nucleic acid is used at a concentration in the range of up to 1 x 10 ^-16 M.   9. A method according to any one of claims 5 to 8, wherein the working electrode comprises an array of nucleic acid capture probes. (a) the electrode system according to claim 1;
(b) means for irradiating the working electrode of the electrode system;
(c) means for applying a potential to the working electrode; and
(d) including means for obtaining a photocurrent action spectrum;
In use, irradiation of the working electrode by means of irradiating the working electrode, and application of a potential to the working electrode by means of applying a potential to the working electrode generates a photocurrent action spectrum, and the photocurrent action spectrum becomes a photocurrent. An apparatus for detecting a target nucleic acid obtained by means for obtaining an action spectrum. (a) the electrode system according to claim 1;
(b) means for irradiating the working electrode of the electrode system;
(c) means for applying a potential to the working electrode; and
(d) including means for obtaining a photocurrent action spectrum;
In use, irradiation of the working electrode by means of irradiating the working electrode, and application of a potential to the working electrode by means of applying a potential to the working electrode generates a photocurrent action spectrum, which is A target nucleic acid quantification apparatus obtained by means for obtaining an action spectrum.   12. Apparatus according to claim 10 or 11, wherein the stable intercalator of the electrode system is a stitched PIND-Ru-PIND bis-intercalator. 13. The device according to any one of claims 10 to 12, wherein the target nucleic acid can be detected or quantified when the target nucleic acid is used at a concentration in the range of up to 1 x ^10-16M .   14. A device according to any one of claims 10 to 13, wherein the working electrode of the electrode system comprises an array of nucleic acid capture probes. (a) chip;
(b) at least one working electrode disposed on the chip;
(c) a nucleic acid capture probe linked to a working electrode, comprising a sequence complementary to the sequence of the target nucleic acid; and
(d) A biosensor chip including a stable intercalator.   16. The biosensor chip according to claim 15, wherein the stable intercalator is a sewn type PIND-Ru-PIND bis-intercalator.   The biosensor chip according to claim 15 or 16, wherein the chip comprises a working electrode.   18. The biosensor chip according to any one of claims 15 to 17, wherein the working electrode comprises an array of nucleic acid capture probes. The biosensor chip according to any one of claims 15 to 18, wherein the target nucleic acid can be detected or quantified when the target nucleic acid is used at a concentration in the range of up to 1 x 10 ^-16 M.

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